A general concept of bispecific reagents is that they provide a physical connection of recombinant entities with at least two binding specificities. The ability to bind two different targets at a high affinity allows greater selectivity in treatment and imaging. For example, Papo et al. (2011) PNAS 108:14067-14072, mutagenized one pole of a single chain vascular endothelial growth factor (VEGF) ligand by introduction of an RGD loop, in order to provide a bispecific reagent that bound both VEGFR2 and αvβ3 integrin. This provides a reagent that binds two different cell surface proteins to achieve a more complete blockage of angiogenesis-associated pathways.
VEGF plays a prominent role in tumor-associated vascular formation. VEGF-mediated signaling is mediated through its interactions with two receptor tyrosine kinases, VEGFR1 (Flt-1) and VEGFR2 (Flk-1 or KDR). VEGFR2, which is expressed in vascular endothelial cells, monocytes, macrophages, and hematopoietic stem cells, is the primary mediator of the mitogenic and angiogenic effects of VEGF. VEGF is a homodimeric ligand that binds two molecules of VEGFR2, one at each pole, thereby triggering receptor dimerization and activation, with a KD of around 100 pM. VEGF-A is the main ligand for VEGFR2, but proteolytically cleaved forms of VEGF-C and VEGF-D may also bind to and activate VEGFR2.
In addition to therapeutic uses, bispecific reagents can provide benefits for molecular imaging. The fundamental challenge for imaging is achieving a high signal over adjacent normal tissues, resulting in high image resolution. In therapy, the challenge is the delivery of tumoricidal doses of the therapeutic agent while sparing normal tissues from unacceptable toxicities. When using antibodies or fragments to target radioactivity to tumors, the binding specificity, pharmacokinetics and biodistribution have to be matched to the radionuclide, tumor, and disease setting for optimal results to be achieved. The nature and size of the immunoglobulin, or its smaller constructs, will determine how quickly it reaches the target antigen and clears from the blood, and the extent, penetration, and duration of its binding to the tumor vs. normal tissues.
Small molecules traditionally used for tumor imaging or therapy suffer from non-specificity. There is therefore a need for reagents that can leverage the advantages of bispecificity and high affinity for its targets, while maintaining an ideal size and stability for tumor targeting. Prostate cancer diagnosis suffers from inaccuracy due to a lack of a single, canonical prostate cancer biomarker. Bispecific antibodies and antibody fragments have been used to address this issue by targeting multiple involved biomarkers to increase specficity, but as tumor imaging tools these molecules are limited by issues of size and stability.
The present invention provides a solution for these issues.